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Acridone derivative 8a induces oxidative stress-mediated apoptosis in CCRF-CEM leukemia cells: application of metabolomics in mechanistic studies of antitumor agents.

Wang Y, Gao D, Chen Z, Li S, Gao C, Cao D, Liu F, Liu H, Jiang Y - PLoS ONE (2013)

Bottom Line: Metabolites from glutathione (GSH) and glycerophospholipid metabolism were investigated in detail, and results showed that GSH level and the reduced/oxidized glutathione (GSH/GSSG) ratio were significantly decreased in 8a-treated cells, while L-cysteinyl-glycine (L-Cys-Gly) and glutamate were greatly increased.In glycerophospholipid metabolism, cell membrane components phosphatidylcholines (PCs) were decreased in 8a-treated cells, while the oxidative products lysophosphatidylcholines (LPCs) were significantly increased.We further found that in 8a-treated cells, the reactive oxygen species (ROS) and lipid peroxidation product malondialdehyde (MDA) were notably increased, accompanied with decrease of mitochondrial transmembrane potential, release of cytochrome C and activation of caspase-3.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Tsinghua University, Beijing, China.

ABSTRACT
A new acridone derivative, 2-aminoacetamido-10-(3, 5-dimethoxy)-benzyl-9(10H)-acridone hydrochloride (named 8a) synthesized in our lab shows potent antitumor activity, but the mechanism of action remains unclear. Herein, we report the use of an UPLC/Q-TOF MS metabolomic approach to study the effects of three compounds with structures optimized step-by-step, 9(10H)-acridone (A), 10-(3,5-dimethoxy)benzyl-9(10H)-acridone (I), and 8a, on CCRF-CEM leukemia cells and to shed new light on the probable antitumor mechanism of 8a. Acquired data were processed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to identify potential biomarkers. Comparing 8a-treated CCRF-CEM leukemia cells with vehicle control (DMSO), 23 distinct metabolites involved in five metabolic pathways were identified. Metabolites from glutathione (GSH) and glycerophospholipid metabolism were investigated in detail, and results showed that GSH level and the reduced/oxidized glutathione (GSH/GSSG) ratio were significantly decreased in 8a-treated cells, while L-cysteinyl-glycine (L-Cys-Gly) and glutamate were greatly increased. In glycerophospholipid metabolism, cell membrane components phosphatidylcholines (PCs) were decreased in 8a-treated cells, while the oxidative products lysophosphatidylcholines (LPCs) were significantly increased. We further found that in 8a-treated cells, the reactive oxygen species (ROS) and lipid peroxidation product malondialdehyde (MDA) were notably increased, accompanied with decrease of mitochondrial transmembrane potential, release of cytochrome C and activation of caspase-3. Taken together our results suggest that the acridone derivative 8a induces oxidative stress-mediated apoptosis in CCRF-CEM leukemia cells. The UPLC/Q-TOF MS based metabolomic approach provides novel insights into the mechanistic studies of antitumor drugs from a point distinct from traditional biological investigations.

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Effects of 8a on the level of MMP and expression of apoptosis-related proteins.(A) MMP affected by 8a in CCRF-CEM cells (n = 3). *p<0.05 compared with vehicle control. (B) Western blot analysis of the effect of 8a on cytochrome C release in CCRF-CEM cells treated with indicated doses of 8a for 24 h. (C) Representative Western blots for the expression of cleaved caspase-3 in CCRF-CEM cells following exposure to different concentrations of 8a for 24 h. β-actin (∼42 kDa) was used as a loading control.
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pone-0063572-g005: Effects of 8a on the level of MMP and expression of apoptosis-related proteins.(A) MMP affected by 8a in CCRF-CEM cells (n = 3). *p<0.05 compared with vehicle control. (B) Western blot analysis of the effect of 8a on cytochrome C release in CCRF-CEM cells treated with indicated doses of 8a for 24 h. (C) Representative Western blots for the expression of cleaved caspase-3 in CCRF-CEM cells following exposure to different concentrations of 8a for 24 h. β-actin (∼42 kDa) was used as a loading control.

Mentions: Mitochondria are an important organelle for cell survival and a vulnerable target of ROS. MMP is important to the integrity and function of mitochondria [46], [47]. To understand the cell death mechanism, we further evaluated MMP in 8a-treated cells using a fluorescent dye Rh123, a cell permeable cationic dye which can specifically binds to the active mitochondria based on the highly negative MMP. As shown in Figure 5A, treatment with 0.5–2 µM 8a for 24 h led to drastic decrease of MMP in a dose-dependent manner. When CCRF-CEM cells were exposed to 0.5, 1.0, and 2.0 µM of 8a, the fold changes of MMP to control cells, calculated from the mean MMP of all tested cells, were 0.837±0.039, 0.556±0.029, and 0.279±0.027, respectively.


Acridone derivative 8a induces oxidative stress-mediated apoptosis in CCRF-CEM leukemia cells: application of metabolomics in mechanistic studies of antitumor agents.

Wang Y, Gao D, Chen Z, Li S, Gao C, Cao D, Liu F, Liu H, Jiang Y - PLoS ONE (2013)

Effects of 8a on the level of MMP and expression of apoptosis-related proteins.(A) MMP affected by 8a in CCRF-CEM cells (n = 3). *p<0.05 compared with vehicle control. (B) Western blot analysis of the effect of 8a on cytochrome C release in CCRF-CEM cells treated with indicated doses of 8a for 24 h. (C) Representative Western blots for the expression of cleaved caspase-3 in CCRF-CEM cells following exposure to different concentrations of 8a for 24 h. β-actin (∼42 kDa) was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646819&req=5

pone-0063572-g005: Effects of 8a on the level of MMP and expression of apoptosis-related proteins.(A) MMP affected by 8a in CCRF-CEM cells (n = 3). *p<0.05 compared with vehicle control. (B) Western blot analysis of the effect of 8a on cytochrome C release in CCRF-CEM cells treated with indicated doses of 8a for 24 h. (C) Representative Western blots for the expression of cleaved caspase-3 in CCRF-CEM cells following exposure to different concentrations of 8a for 24 h. β-actin (∼42 kDa) was used as a loading control.
Mentions: Mitochondria are an important organelle for cell survival and a vulnerable target of ROS. MMP is important to the integrity and function of mitochondria [46], [47]. To understand the cell death mechanism, we further evaluated MMP in 8a-treated cells using a fluorescent dye Rh123, a cell permeable cationic dye which can specifically binds to the active mitochondria based on the highly negative MMP. As shown in Figure 5A, treatment with 0.5–2 µM 8a for 24 h led to drastic decrease of MMP in a dose-dependent manner. When CCRF-CEM cells were exposed to 0.5, 1.0, and 2.0 µM of 8a, the fold changes of MMP to control cells, calculated from the mean MMP of all tested cells, were 0.837±0.039, 0.556±0.029, and 0.279±0.027, respectively.

Bottom Line: Metabolites from glutathione (GSH) and glycerophospholipid metabolism were investigated in detail, and results showed that GSH level and the reduced/oxidized glutathione (GSH/GSSG) ratio were significantly decreased in 8a-treated cells, while L-cysteinyl-glycine (L-Cys-Gly) and glutamate were greatly increased.In glycerophospholipid metabolism, cell membrane components phosphatidylcholines (PCs) were decreased in 8a-treated cells, while the oxidative products lysophosphatidylcholines (LPCs) were significantly increased.We further found that in 8a-treated cells, the reactive oxygen species (ROS) and lipid peroxidation product malondialdehyde (MDA) were notably increased, accompanied with decrease of mitochondrial transmembrane potential, release of cytochrome C and activation of caspase-3.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Tsinghua University, Beijing, China.

ABSTRACT
A new acridone derivative, 2-aminoacetamido-10-(3, 5-dimethoxy)-benzyl-9(10H)-acridone hydrochloride (named 8a) synthesized in our lab shows potent antitumor activity, but the mechanism of action remains unclear. Herein, we report the use of an UPLC/Q-TOF MS metabolomic approach to study the effects of three compounds with structures optimized step-by-step, 9(10H)-acridone (A), 10-(3,5-dimethoxy)benzyl-9(10H)-acridone (I), and 8a, on CCRF-CEM leukemia cells and to shed new light on the probable antitumor mechanism of 8a. Acquired data were processed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to identify potential biomarkers. Comparing 8a-treated CCRF-CEM leukemia cells with vehicle control (DMSO), 23 distinct metabolites involved in five metabolic pathways were identified. Metabolites from glutathione (GSH) and glycerophospholipid metabolism were investigated in detail, and results showed that GSH level and the reduced/oxidized glutathione (GSH/GSSG) ratio were significantly decreased in 8a-treated cells, while L-cysteinyl-glycine (L-Cys-Gly) and glutamate were greatly increased. In glycerophospholipid metabolism, cell membrane components phosphatidylcholines (PCs) were decreased in 8a-treated cells, while the oxidative products lysophosphatidylcholines (LPCs) were significantly increased. We further found that in 8a-treated cells, the reactive oxygen species (ROS) and lipid peroxidation product malondialdehyde (MDA) were notably increased, accompanied with decrease of mitochondrial transmembrane potential, release of cytochrome C and activation of caspase-3. Taken together our results suggest that the acridone derivative 8a induces oxidative stress-mediated apoptosis in CCRF-CEM leukemia cells. The UPLC/Q-TOF MS based metabolomic approach provides novel insights into the mechanistic studies of antitumor drugs from a point distinct from traditional biological investigations.

Show MeSH
Related in: MedlinePlus